A hallmark of virtually every cancer is aberrant epigenetic silencing of vital genes required for a normal cell cycle and genome stability (e.g. MLH1, BRCA1, VHL, p16, etc.). Loss of expression of these tumor suppressor genes plays a critical role in cancer formation and growth. Thus, targeting this root cause of cancer has significant potential to treat and potentially cure a host of cancers. Indeed, a major initiative i essentially every major pharmaceutical company is to investigate epigenetic cancer therapies targeting the reactivation of these key genes. However, despite its promise, at this time drug-induced gene reactivation does not work therapeutically because reactivation is invariably unstable. One reason for this failure is that current approaches consider silencing as an end point instead of a process, and as a result do not strategically target epigenetic modifications that occur at different times during the silencing process. We believe achieving stable reactivation of tumor suppressor genes requires not only targeting the end points of silencing (e.g., DNA methylation), but also targeting the early steps of the silencing process. Nzumbe's long-term goal is to develop screening assays using our patent pending platform to induce epigenetic silencing and then identify compounds that inhibit the start of the silencing process. No other company has the ability to perform this type of screen. For this Phase I application, we will develop screens for two clinically relevant tumor suppressor genes that undergo epigenetic silencing in human cancers; BRCA1, which is silenced in breast cancer, and MLH1, which is silenced in colon cancer. Combined these two cancers account for approximately 370,000 new patients and 90,000 deaths every year. Briefly, our primary screen will involve expressing green fluorescent protein (GFP) and luciferase (Luc) from the MLH1 and BRCA1 promoters, inducing gene silencing, and then measuring changes in fluorescence intensity in microtiter plates that will indicate whether test compounds exhibit inhibitory activities. Two specific aims are proposed. For the first aim, we will develop the BRCA1 and MLH1 screens in breast and colon cancer cells, respectively, and calibrate the assays to ensure reproducibility and low levels of variability. For the second aim, we validate the screening assays, first with known positive and negative controls and then with a set of 11 test compounds exhibiting known and distinct epigenetic modifying activities. Successful completion of the proposed work will provide commercially viable assays to screen small and large molecule libraries. Ultimately, compounds identified with the BRCA1 and MLH1 assays can be used as part of treatment regimens for breast and colon cancer. Because our approach can be modified for a variety of tumor suppressor gene promoters, we will propose in a Phase II application to develop a highly desirable and commercially viable drug screening service that is applicable to a variety of cancers